Keratocyte density: comparison of two confocal microscopes

The ConfoScan 3 clinical confocal microscope provides sharper images of cells in the stroma than the Tandem Scanning confocal microscope does. In this study, we compared volumetric densities of stromal cells determined from images recorded by these two instruments.

Fifty corneas of 25 normal subjects were examined by confocal microscopy, first by using a Tandem Scanning confocal microscope and then by using a ConfoScan 3 confocal microscope. Bright objects, assumed to represent keratocytes, were counted in a known area of two frames selected from the mid-stroma. The effective depth of the sample volume represented by each frame was estimated from the number of consecutive frames and the corresponding distance that selected cells were visible and countable during a scan. Density was the number of visible cells in the sample area divided by the effective sample volume.

The effective focal depth of the Tandem Scanning microscope was 11.9 +/- 2.6 microm (mean +/- SD), and was 25.9 +/- 7.1 microm for the ConfoScan 3. Mean cell density at mid stroma was 23,013 +/- 4,420 cells/mm(3) with the Tandem Scanning microscope and 23,996 +/- 2,898 cells/mm(3) with the ConfoScan 3. This difference was not significant (P = 0.15).

In normal corneas, the ConfoScan 3 and the Tandem Scanning confocal microscopes indicate stromal cell densities that are not significantly different from each other. Estimates of cell density from both instruments require an accurate estimate of the effective depth of the sample volume; this depth is approximately 2.2 times greater with the ConfoScan 3. This difference must be considered when comparing results from studies that use one instrument with results from studies that use the other.